We have previously shown that binding of LFA-1 on NK cells to ICAM-1 on target cells is sufficient to induce the accumulation of perforin-containing cytotoxic granules and the microtubule organizing center (MTOC) at the site of NK cell contact with the target cell. Cytotoxicity occurs when this polarization signal is combined with a second signal that triggers degranulation. This behavior contrasts with that of LFA-1 in T cells, which requires "inside-out" signaling for its own activation. In T cells, perforin polarization requires engagement of the T cell antigen receptor (TCR), and costimulation with LFA-1 greatly enhances polarization of perforin-containing granules. We investigated whether LFA-1 in NK cells signals through immunoreceptor tyrosine-based activation motif (ITAM)-containing adapter proteins, such as DAP12 and FcR &#947;chain, and if molecules such as paxillin are involved in LFA-1-induced perforin polarization. In this report, we demonstrate that beta2 integrin engagement by ICAM-1 alone leads to tyrosine phosphorylation of the TCR zeta chain, Syk, and PLC-gamma1 and PLC-gamma2 in human NK cells. Inhibition of Syk and PLC-gamma enzymatic activity prevents perforin polarization in response to ICAM-1 expressed on target cells. Comparison to a known ITAM-dependent signaling pathway in NK cells, the CD16 response, reveals surprising similarity in the proximal biochemical responses, despite very different downstream outcomes with regard to perforin polarization and degranulation. Further investigation reveals differential requirement of the adapter proteins paxillin and LAT for responses to LFA-1 and CD16, suggesting the importance of different signaling complex assemblies or compartmentalization of signaling molecules to determine functional outcomes. Binding of &#946;2 integrin LFA-1 to ICAM is sufficient to induce granule polarization but not degranulation, whereas CD16 binding to IgG triggers unpolarized degranulation. We have investigated here the basis for this difference. IL-2 expanded human NK cells were stimulated by incubation with plate-bound ligands of LFA-1 (ICAM-1) and CD16 (human IgG). Surprisingly, LFA-1 elicited signals similar to those induced by CD16, including tyrosine phosphorylation of the TCR zeta chain, tyrosine kinase Syk, and phospholipase C (PLC)-gamma. Whereas CD16 activated Ca2+ mobilization and LAT phosphorylation, LFA-1 did not, but induced strong Pyk2 and paxillin phosphorylation. LFA-1-dependent granule polarization was blocked by inhibition of Syk, PLC-gamma, and PKC, and by paxillin knockdown. Therefore, common signals triggered by CD16 and LFA-1 bifurcate to provide independent control of Ca2+-dependent degranulation and paxillin-dependent granule polarization. To further explore the pathway by which LFA-1 signals to induce granule and MTOC polarization towards the immunological synapse in NK cells, we have employed a mass spectrometry approach. Primary NK cells stimulated by purified, recombinant ICAM-1 bound to plates were lysed, and tyrosine-phosphorylated proteins were isolated with a phosphotyrosine-specific antibody. As control, NK cells were also added to plates without ICAM-1. For comparison, NK cells were similarly stimulated by CD16 on plates carrying human IgG1. Mass spectrometry was then applied to sequence all the proteins isolated in this manner, which includes tyrosine-phosphorylated proteins and proteins bound to complexes that include tyrosine-phosphorylated proteins. This approach identified many proteins, some of which were enriched specifically in the ICAM-1-stimulated NK cells. The approach was validated by the presence of several proteins that had already been identified as undergoing tyrosine phosphorylation, such as Syk, PLC-gamma, paxillin, p130CAS, and others. An additional set of proteins, was identified, which was specific to stimulation through LFA-1. Using siRNA-mediated knock-down of expression and functional assays for adhesion and granule polarization we have identified several molecules that contribute to the LFA-1-dependent signal for polarization.